Dataset Information

Identification of an arabinopyranosyltransferase from Physcomitrella patens involved in the synthesis of the hemicellulose xyloglucan.

ABSTRACT: The hemicellulose xyloglucan consists of a backbone of a ?-1,4 glucan substituted with xylosyl moieties and many other, diverse side chains that are important for its proper function. Many, but not all glycosyltransferases involved in the biosynthesis of xyloglucan have been identified. Here, we report the identification of an hitherto elusive xyloglucan:arabinopyranosyltransferase. This glycosyltransferase was isolated from the moss Physcomitrella patens, where it acts as a xyloglucan "D"-side chain transferase (XDT). Heterologous expression of PpXDT in the Arabidopsis thaliana double mutant mur3.1 xlt2, where xyloglucan consists of a xylosylated glucan without further glycosyl substituents, results in the production of the arabinopyranose-containing "D" side chain as characterized by oligosaccharide mass profiling, glycosidic linkage analysis, and NMR analysis. In addition, expression of a related Physcomitrella glycosyltransferase ortholog of PpXLT2 leads to the production of the galactose-containing "L" side chain. The presence of the "D" and "L" xyloglucan side chains in the Arabidopsis double mutant Atmur3.1 xlt2 expressing PpXDT and PpXLT2, respectively, rescues the dwarfed phenotype of untransformed Atmur3.1 xlt2 mutants to nearly wild-type height. Expression of PpXDT and PpXLT2 in the Atmur3.1 xlt2 mutant also enhanced root growth.

SUBMITTER: Zhu L

PROVIDER: S-EPMC6508525 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification of an arabinopyranosyltransferase from <i>Physcomitrella patens</i> involved in the synthesis of the hemicellulose xyloglucan.

Zhu Lei L Dama Murali M Pauly Markus M

Plant direct 20180313 3

The hemicellulose xyloglucan consists of a backbone of a β-1,4 glucan substituted with xylosyl moieties and many other, diverse side chains that are important for its proper function. Many, but not all glycosyltransferases involved in the biosynthesis of xyloglucan have been identified. Here, we report the identification of an hitherto elusive xyloglucan:arabinopyranosyltransferase. This glycosyltransferase was isolated from the moss <i>Physcomitrella patens</i>, where it acts as a xyloglucan "D ...[more]

PMID: 31245712

Similar Datasets

Project description:BACKGROUND: Alternative splicing (AS) involving tandem acceptors that are separated by three nucleotides (NAGNAG) is an evolutionarily widespread class of AS, which is well studied in Homo sapiens (human) and Mus musculus (mouse). It has also been shown to be common in the model seed plants Arabidopsis thaliana and Oryza sativa (rice). In one of the first studies involving sequence-based prediction of AS in plants, we performed a genome-wide identification and characterization of NAGNAG AS in the model plant Physcomitrella patens, a moss. RESULTS: Using Sanger data, we found 295 alternatively used NAGNAG acceptors in P. patens. Using 31 features and training and test datasets of constitutive and alternative NAGNAGs, we trained a classifier to predict the splicing outcome at NAGNAG tandem splice sites (alternative splicing, constitutive at the first acceptor, or constitutive at the second acceptor). Our classifier achieved a balanced specificity and sensitivity of >or= 89%. Subsequently, a classifier trained exclusively on data well supported by transcript evidence was used to make genome-wide predictions of NAGNAG splicing outcomes. By generation of more transcript evidence from a next-generation sequencing platform (Roche 454), we found additional evidence for NAGNAG AS, with altogether 664 alternative NAGNAGs being detected in P. patens using all currently available transcript evidence. The 454 data also enabled us to validate the predictions of the classifier, with 64% (80/125) of the well-supported cases of AS being predicted correctly. CONCLUSION: NAGNAG AS is just as common in the moss P. patens as it is in the seed plants A. thaliana and O. sativa (but not conserved on the level of orthologous introns), and can be predicted with high accuracy. The most informative features are the nucleotides in the NAGNAG and in its immediate vicinity, along with the splice sites scores, as found earlier for NAGNAG AS in animals. Our results suggest that the mechanism behind NAGNAG AS in plants is similar to that in animals and is largely dependent on the splice site and its immediate neighborhood.

Project description:BackgroundThe moss Physcomitrella patens contains C18- as well as C20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2.ResultsOur results show that PpAOS1 is highly active with both C18 and C20-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C20-substrates, exhibiting trace activity (~1000-fold lower kcat/KM) with C18 substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS - a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced cis(+)-12-oxo phytodienoic acid accumulation in PpAOS1 and PpAOS2 single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased cis(+)-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither PpAOS1 nor PpAOS2 showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis.ConclusionsOur study highlights five findings regarding the oxylipin metabolism in P. patens: (i) Both AOS isoforms are capable of metabolizing C18- and C20-derived substrates with different specificities suggesting that both enzymes might have different functions. (ii) Site directed mutagenesis demonstrated that the catalytic trajectories of 9-/13-PpAOS1 and PpHPL are closely inter-connected and PpAOS1 can be inter-converted by a single amino acid exchange into a HPL. (iii) In contrast to PpAOS1, PpAOS2 is localized in the plastid where oxylipin metabolism takes place. (iv) PpAOS1 is essential for wound-induced accumulation of cis(+)-12-oxo phytodienoic acid while PpAOS2 appears not to be involved in the process. (v) Knock-out mutants of neither AOS showed a deviating morphological phenotype suggesting that there are overlapping functions with other Cyp74 enzymes.

Dataset Information

Identification of an arabinopyranosyltransferase from Physcomitrella patens involved in the synthesis of the hemicellulose xyloglucan.

Publications

Identification of an arabinopyranosyltransferase from <i>Physcomitrella patens</i> involved in the synthesis of the hemicellulose xyloglucan.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure